The Center for Fluorescence Spectroscopy (CFS) is a multi-user facility which is focused on the advanced biomedical applications of fluorescence. The CFS was first supported by the National Center for Research Resources in April of 1993 when it received four years of funding. In this competitive renewal request we propose development of the following core technologies: 1. Development of long-lifetime (100 ns to 5 mus) and love-wavelength probes for use in fluorescence spectroscopy, microscopy and sensing. We will emphasize development of metal-ligand complexes which are conjugatable to macromolecules, or which are sensitive to Ca2+, Mg2+, K+, Na+ and pH, and which display high quantum yields, analyte-sensitive lifetimes, and/or high anisotropy. 2. Two- and three-photon excitation of biochemical fluorophores, characterization of decay times, photostability, excitation and anisotropy spectra, and studies of alkane fluorescence with two-photon excitation. 3.Development of the experimental and theoretical aspects of light quenching, which allows control of the excited state population by long wavelength light pulses. 4. Development of the methodology of fluorescence lifetime imaging microscopy, including software and instrumentation, using xenopus embryos, cos, PC12, 293 and neonatal heart cells as model systems. 5. Development of cell-by-cell lifetime measurements of intracellular ions in flow cytometry. 6. Continued development of software for time domain (TD), frequency- domain (FD) and FLIM. The CFS will continue to work with collaborators while developing the core technologies, and to provide access to TD and TD measurement for many internal and external collaborators and users. Dissemination of information is accomplished by the presence of our CFS booth at national meetings, organization of bi-annual national meetings, the Journal of Fluorescence and the newly founded Journal of Biomedical Optics. Additionally, each year the CFS teaches an intensive week-long course on the principles of time resolved fluorescence.